Purpose:
Autophagy, an essential homeostatic process, is an important pro-survival mechanism involving the lysosomal degradation pathway in response to various signals, including dangerous stimuli such as infection. The role of autophagy and its potential regulation by TLR4, which is higher in C57BL/6 (B6) over BALB/c mice during Pseudomonas aeruginosa induced keratitis, is unknown and the purpose of this study.

Methods:
Wild type (WT) B6, BALB/c (with or without rapamycin treatment to induce autophagy) and TLR4 functionally deficient, C.C3-Tlr4lps-d (Tlr4lps-d) mice were infected and tested using various approaches including: a Mouse Autophagy RT2 ProfilerTM PCR array (containing 84 autophagy related genes), real time RT-PCR, and immunostaining (elastase and MPO) for neutrophil extracellular traps (NETs). In vitro, primary cultured corneal epithelium from WT B6 and BALB/c mice (confluent, 2nd passage cells) also was treated with ultrapure LPS (only activates TLR4 pathway; 1μg/ml for 18h) and tested by a PCR array, as above. Other, separate in vitro experiments were similarly done with or without the addition of rapamycin (1μM for 18 h). For both of the above in vitro experiments, real time RT-PCR was used to further evaluate autophagy markers.

Results:
PCR array (3 days p.i.) and real-time RT-PCR (1-5 days p.i.) demonstrated that several pro-autophagy genes (beclin-1, LC3A, LC3B, and IRGM1) were up-regulated, while autophagy degradation molecules (p62 and NBR1) were down-regulated in the cornea of B6 vs. BALB/c mice. Pro-inflammatory NETs, associated with autophagy-related signaling, also were more prominent and seen earlier in B6 vs. BALB/c mice. In vivo rapamycin treated BALB/c mice showed enhanced levels of TLR4 (and IFN-Υ) and a similar pattern for the above autophagy and degradation molecule related genes over controls. Conversely, RT-PCR showed that Tlr4lps-d vs. WT BALB/c mice had decreased mRNA levels of beclin-1, LC3A, LC3B, and IRGM1, while p62 and NBR1 were increased. In vitro PCR array studies revealed numerous (23) autophagy genes in the epithelium of B6 vs. BALB/c mice that were changed (two-fold or greater) and these were selectively confirmed by RT-PCR.

Conclusions:
These data provide evidence that autophagy is important in keratitis, is associated with NETs and that TLR4 appears to contribute to its regulation.